Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus

被引：70

作者：

Bansal, S. ^{[1
]}

Gnaneswari, D. ^{[1
]}

Mishra, P. ^{[1
]}

Kundu, B. ^{[1
,2
]}

机构：

[1] Indian Inst Technol Delhi, Dept Biochem Engn & Biotechnol, New Delhi 110016, India

[2] Indian Inst Technol Delhi, Sch Biol Sci, New Delhi 110016, India

来源：

BIOCHEMISTRY-MOSCOW | 2010年 / 75卷 / 03期

关键词：

Pyrococcus furiosus; L-asparaginase; hyperthermophile; chemical denaturation; homology model; stability; enzyme kinetics; ESCHERICHIA-COLI ASPARAGINASE; ACRYLAMIDE FORMATION; FLUOROMETRIC ASSAY; ERWINIA-CAROTOVORA; POTATO PRODUCTS; PURIFICATION; REDUCTION; GLYCOSYLASPARAGINASE; BIOTECHNOLOGY; EXPRESSION;

D O I：

10.1134/S0006297910030144

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K (m) of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.

引用

页码：375 / 381

页数：7

共 40 条

[1] Acrylamide in gingerbread:: Critical factors for formation and possible ways for reduction [J].

Amrein, TM ;

Schönbächler, B ;

Escher, F ;

Amadó, R .

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 2004, 52 (13) :4282-4288

[2]

ASHWORTH LA, 1974, CANCER RES, V34, P1353

[3] Dynamics of a mobile loop at the active site of Escherichia coli asparaginase [J].

Aung, HP ;

Bocola, M ;

Schleper, S ;

Röhm, KH .

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 2000, 1481 (02) :349-359

[4]

BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3

[5] Helicobacter pylori L-asparaginase: A promising chemotherapeutic agent [J].

Cappelletti, Donata ;

Chiarelli, Laurent R. ;

Pasquetto, Maria Valentina ;

Stivala, Simona ;

Valentini, Giovanna ;

Scotti, Claudia .

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2008, 377 (04) :1222-1226

[6] ARTIFICIAL CELLS IN MEDICINE AND BIOTECHNOLOGY [J].

CHANG, TMS .

APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 1984, 10 :5-24

[7]

Ciesarová Z, 2006, J FOOD NUTR RES-SLOV, V45, P141

[8] A SPECIFIC L-ASPARAGINASE FROM THERMUS-AQUATICUS [J].

CURRAN, MP ;

DANIEL, RM ;

GUY, GR ;

MORGAN, HW .

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1985, 241 (02) :571-576

[9] Engineering the substrate specificity of Escherichia coli asparaginase II.: Selective reduction of glutaminase activity by amino acid replacements at position 248 [J].

Derst, C ;

Henseling, J ;

Röhm, KH .

PROTEIN SCIENCE, 2000, 9 (10) :2009-2017

[10] STATES AND FUNCTIONS OF TYROSINE RESIDUES IN ESCHERICHIA-COLI ASPARAGINASE-II [J].

DERST, C ;

WEHNER, A ;

SPECHT, V ;

ROHM, KH .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1994, 224 (02) :533-540

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